Purpose.:
To investigate the roles of the CCL2-CCR2 and CX3CL1-CX3CR1 pathways in experimental autoimmune uveoretinitis (EAU)-mediated retinal tissue damage and angiogenesis.

Methods.:
The C57BL/6J wild-type (WT) and CCL2−/−CX3CR1gfp/gfp (double knockout [DKO]) mice were immunized with IRBP1-20. Retinal inflammation and tissue damage were evaluated clinically and histologically at different days postimmunization (p.i.). Retinal neovascular membranes were evaluated by confocal microscopy of retinal flat mounts, and immune cell infiltration by flow cytometry.

Results.:
At day 25 p.i., DKO mice had lower clinical and histological scores and fewer CD45highCD11b+ infiltrating cells compared with WT mice. The F4/80+ macrophages constitute 40% and 21% and CD11b+Gr-1+Ly6G+ neutrophils constitute 10% and 22% of retinal infiltrating cells in WT and DKO mice, respectively. At the late stages of EAU (day 60–90 p.i.), DKO and WT mice had similar levels of inflammatory score. However, less structural damage and reduced angiogenesis were detected in DKO mice. Neutrophils were rarely detected in the inflamed retina in both WT and DKO mice. Macrophages and myeloid-derived suppressor cells (MDSCs) accounted for 8% and 3% in DKO EAU retina, and 19% and 10% in WT EAU retina; 71% of infiltrating cells were T/B-lymphocytes in DKO EAU retina and 50% in WT EAU retina.

Conclusions.:
Experimental autoimmune uveoretinitis–mediated retinal tissue damage and angiogenesis is reduced in CCL2−/−CX3CR1gfp/gfp mice. Retinal inflammation is dominated by neutrophils at the acute stage and lymphocytes at the chronic stage in these mice. Our results suggest that CCR2+ and CX3CR1+ monocytes are both involved in tissue damage and angiogenesis in EAU.

Introduction

Chemokines and chemokine receptors are essential for homing of immune cells under patho-physiological conditions. Chemokines are classified into two functional groups: homeostatic chemokines that are constitutively produced in certain tissues and are responsible for basal leukocyte migration, and inflammatory chemokines that are produced under pathological conditions and are essential for migration of immune cells into the site of inflammation. In the retina, chemokine CX3CL1 (also known as fractalkine in humans) is constitutively produced by retinal neurons1 and its receptor CX3CR1 is exclusively expressed by microglia.2 The CX3CL1-CX3CR1 pathway is known to negatively regulate microglial activation under patho-physiological conditions.2 The CX3CL1 also is an important cytokine for homing of CX3CR1+ monocytes to tissues.3 Chemokine CCL2 is a typical inflammatory cytokine. Many retinal cells, including retinal pigment epithelia (RPE) and microglia can produce large amounts of CCL2 under inflammatory conditions.4,5 The CCL2 is crucial for the recruitment of CCR2+ monocytes to site of inflammation.

The role of the CX3CL1-CX3CR1 and CCL2-CCR2 pathways in retinal health and disease has been studied extensively in the past decade. Although the CX3CL1-CX3CR1 pathway critically controls microglial activation, deletion of CX3CR1 does not affect the recruitment and distribution of retinal microglia under normal physiological conditions.6 We have shown that the retinal microglia can be replaced by bone marrow–derived myeloid cells within 6 months after whole-body irradiation,7 and the recruitment of these cells is CCL2-CCR2 but not CX3CL1-CX3CR1 pathway dependent.8 However, under aging conditions, it has been reported that CX3CR1 KO mice9 and CCL2 KO or CCR2 KO10,11 mice develop atrophic lesions akin to human AMD. Although the early onset of retinal lesions in the CCL2/CX3CR1 double knockout (DKO) mice reported by Tuo et al.12 has been shown to be related to rd8 mutation in the crumbs1 gene,13 we observed age- and light-dependent development of localized retinal atrophies in the CCL2−/−CX3CR1gfp/gfp mice that do not carry the rd8 mutation,14 suggesting that CCL2-CCR2 and CX3CL1-CX3CR1 pathways are involved in retinal defence under chronic stress conditions.

The role of the CCL2-CCR2 and CX3CL1-CX3CR1 pathways in retinal autoimmune response has been investigated in experimental autoimmune uveoretinitis (EAU), a model for human noninfectious posterior uveitis.15 Although macrophage recruitment is impaired in CCL2 KO or CCR2 KO EAU mice, the severity of inflammation remains unchanged.16 Further studies suggest that retinal inflammation in CCL2 KO or CCR2 KO EAU mice16,17 is dominated by neutrophils. The deletion of CX3CR1 does not affect EAU,18 although one study reported increased disease severity in CX3CR1 KO EAU mice.19 Furthermore, a recent study by London et al.20 suggests that CX3CR1hi infiltrating macrophages are involved in the resolution of inflammation, and the CCL2-CCR2 pathway is required for the recruitment of myeloid-derived suppressor cells (MDSCs).20 These data suggest that both CCL2-CCR2 and CX3CL1-CX3CR1 pathways may be involved in the late stages of inflammation in EAU.

We have shown that EAU in wild-type (WT) C57BL/6J mice does not spontaneously resolve, and chronic inflammation persists for more than 4 months.17 We also have shown that chronic inflammation induces retinal angiogenesis,17 which is driven by CCL2 ligation.17 The roles of the CCL2-CCR2 and CX3CL1-CX3CR1 pathways in EAU, in particular, during the chronic stage warrant further investigation.

In this study, we show that the severity of inflammation is reduced in CCL2−/−CX3CR1gfp/gfp mice during the acute stage but not the chronic stage. Despite similar levels of inflammation at the late stages of EAU, retinal structural damage and angiogenesis are reduced in the CCL2−/−CX3CR1gfp/gfp mice. Retinal inflammation in CCL2−/−CX3CR1gfp/gfp mice is dominated by neutrophils at the acute stage and lymphocytes at the late stages of EAU.

Methods

Animals

All mice used in this study were 2 to 3 months old at the time of EAU induction. The C57BL/6J mice were used as WT controls. The CCL2−/−CX3CR1gfp/gfp (DKO) mice were generated using CCL2−/− mice (B6.129S-Ccltm1R°l/J; Jackson Laboratory, Bar Harbor, ME, USA) and CX3CR1gfp/gfp mice21; described previously,14 DNA sequencing confirmed that the mice do not carry the rd8 mutation in the crumbs1 gene.14 All mice were maintained in the Biological Research Unit (BRU) at Queen's University Belfast, United Kingdom. All in vivo procedures were conducted under the regulation of the UK Home Office Animals (Scientific Procedures) Act 1986, and the study was in compliance with the ARVO Statement for the Use of Animals in Ophthalmology and Vision Research.

Mouse eyes were enucleated on days 25 and 90 post immunization (p.i.) and fixed in 2.5% (wt/vol) glutaraldehyde (Agar Scientific Ltd., Stansted, UK) for at least 24 hours. Eyes were then embedded in paraffin and processed for hematoxylin and eosin (H&E) staining. For each eye, four sections from four different layers were graded. The infiltrating and structural scores of retinal inflammation were graded using the criteria described previously.24

Flow Cytometry

Preparation of Cells From Mice Blood and Tissues.

Mice were killed by CO2 inhalation and blood collected by cardiac puncture; 100 μL blood was used for FACS staining. The spleens were homogenized and passed through a 100-μm cell strainer (BD Labware, Oxford, UK) to obtain single-cell suspension. Red blood cells were removed with lysis buffer and 2 × 105 splenocytes were used for FACS staining. The eyes were enucleated, and the retinas were dissected in cold PBS. The retinas were then treated with 1 mg/mL collagenase I (Sigma-Aldrich, Dorset, UK) at 37°C for 30 minutes. The single-cell suspension was washed and filtered through a 100-μm cell strainer (BD Labware). The cell suspension from one retina was used for each FACS staining.

FACS Staining and Acquisition.

After blocking the Fcγ receptor, the cells were incubated with 50 μL fluorochrome-conjugated antibody cocktail (Table 1) for 40 minutes on ice. The samples were washed with FACS buffer, re-suspended in 200 μL FACS buffer, and processed for FACS analysis using the BD FACSCantoII (BD Biosciences, Oxford, UK). The data were subsequently analyzed by FlowJo Software (TreeStar, Inc., Ashland, OR, USA). To quantify the cell number in the retinas, all samples were run at a constant flow speed. The cell number (N) is calculated by N = T/t × n. T is the time required to run 200 μL FACS buffer, t is the time used for each sample, and n is the cell number acquired.

The total RNAs of retinal samples were extracted by RNeasy Mini Kit (Qiagen, West Sussex, UK) according to the manufacturer's instructions; 1 μg total RNA was used for reverse transcription using the SuperScript II Reverse Transcriptase kit (Invitrogen, Paisley, UK) according to the manufacturer's instructions. Murine mRNA expression levels were quantified by real-time PCR using the LightCycler 480 system with SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany). The primers used are listed in Table 2; 18S was used as a housing keeping gene. Gene fold changes were calculated by dividing the normalized values of EAU/DKO nonimmunized samples by normalized values of WT nonimmunized samples.

Experimental autoimmune uveoretinitis clinical and histological scores were compared by Mann-Whitney U test. Angiogenesis data of WT and DKO mice were analyzed using an unpaired two-tailed Student's t-test. The immune cell subsets, and immune-related gene expression in WT and DKO mice at different stages of EAU were analyzed by two-way ANOVA. All data were presented as mean ± SEM. Probability values of less than 0.05 were considered as statistically significant.

Histological examination revealed a considerable number of infiltrating cells in the vitreous cavity and retina in day 25 p.i. WT mice (Fig. 2A). Severe vasculitis (arrow, Fig. 2A) and retinal folds were frequently observed in WT EAU mice (arrowhead, Fig. 2A). Fewer infiltrating cells and mild vasculitis were observed in DKO EAU mice (Fig. 2B). Both the infiltration score and structural damage score were significantly lower in DKO mice compared with those in WT mice (Fig. 2C). At day 90 p.i., photoreceptor outer segment was largely absent in WT EAU mice (Fig. 2D). Granuloma and large scars also were frequently observed (Fig. 2D). In some cases, severe degeneration of the retina and the total loss of photoreceptors were observed (Fig. 2E). In DKO EAU mice, few infiltrating cells were observed in the retina (Fig. 2F), and the retinal layers remained intact (Fig. 2F). Large granulomas and scar lesions were rarely observed (Fig. 2F), although photoreceptor outer segment damage was observed in some mice (Fig. 2G). The structural damage score was significantly lower in DKO mice compared with WT EAU mice (Fig. 2H), although the infiltrative score between WT and DKO EAU mice did not significantly differ (Fig. 2H).

Previous studies have shown that the severity of EAU was not affected in CCL2 KO16 or CX3CR1 KO18 mice, and this was further confirmed in this study (Supplementary Fig. S1). Our results suggest that the combined deletion of CCL2 and CX3CR1 protects the retina from EAU-mediated inflammatory damage.

Retinal Angiogenesis in Late Stages of EAU in WT and CCL2−/−CX3CR1gfp/gfp Mice

Kinetics of Circulating Immune Cells During Different Stages of EAU in WT and CCL2−/−CX3CR1gfp/gfp EAU Mice

Previously we have shown that there is no difference in different subsets of immune cells in the blood and spleen between WT and DKO mice at different ages.17 In this study, we also observed no difference between nonimmunized WT and DKO mice (Figs. 4A, 4C).

Flow cytometric analysis of immune cells in the blood and spleen of WT and DKO mice. Leukocytes from blood and spleen of WT and DKO mice were analyzed by flow cytometry for the presence of various immune cells. (A) Bar graph showing the percentages of various immune cells in the blood in nonimmunized WT and DKO mice. (B) The percentage of different subsets of immune cells in the blood at different stages of EAU in WT and DKO mice. (C) Bar graph showing the percentages of various immune cells in the splenic in nonimmunized WT and DKO mice. (D) the percentage of different subsets of immune cells in the spleen at different stages of EAU. Data were presented as mean ± SEM. n = 3. *P < 0.05, **P < 0.01 compared with nonimmunized mice. +P < 0.05 , ++P < 0.01 compared with WT mice of the same time point.

Figure 4

Flow cytometric analysis of immune cells in the blood and spleen of WT and DKO mice. Leukocytes from blood and spleen of WT and DKO mice were analyzed by flow cytometry for the presence of various immune cells. (A) Bar graph showing the percentages of various immune cells in the blood in nonimmunized WT and DKO mice. (B) The percentage of different subsets of immune cells in the blood at different stages of EAU in WT and DKO mice. (C) Bar graph showing the percentages of various immune cells in the splenic in nonimmunized WT and DKO mice. (D) the percentage of different subsets of immune cells in the spleen at different stages of EAU. Data were presented as mean ± SEM. n = 3. *P < 0.05, **P < 0.01 compared with nonimmunized mice. +P < 0.05 , ++P < 0.01 compared with WT mice of the same time point.

After EAU induction, WT mice experienced increased proportions of CD11b+ cells, Ly6G+ neutrophils, and F4/80+ macrophages in the blood at day 25 p.i. (Fig. 4B). By day 90 p.i., the proportion of Ly6G+ neutrophils had returned to basal levels, although CD11b+ and F4/80+ cells were higher than those in basal levels (i.e., nonimmunized mice) (Fig. 4B). Interestingly, the proportion of CD8+ cells decreased significantly in EAU mice compared with nonimmunized controls (Fig. 4B). The proportion of CD4+ cells and CD11b+Gr-1+Ly6G− (i.e., MDSCs) remained unchanged in the blood at day 25 p.i. (Fig. 4B). By day 90 p.i., the proportion of CD4+ cells reduced and MDSCs increased compared with that in day 25 p.i. (Fig. 4B). The dynamic change of different subsets of blood leukocytes in DKO EAU mice was similar to that in WT mice apart from F4/80+ macrophages, which only slightly increased at day 25 p.i. in DKO mice (Fig. 4B).

The dynamic change of a different subset of immune cells in the spleen during EAU is similar to that in the blood in WT and DKO mice (Fig. 4D). Double knockout mice had significantly lower levels of F4/80+ macrophages compared with WT mice at day 90 p.i. (Fig. 4D).

The CD11b+CD45int cells are known to be resident microglial cells and the CD45hi cells are known to be infiltrating leukocytes.26 Flow cytometry analysis confirmed that the CD11b+CD45int microglial cells (Fig. 5A) were negative for Ly6G, Gr-1, CD4, and CD8 (Fig. 5B, G1 cells), and the CD45hi cells (Fig. 5A) include Ly6G+, Gr-1+, CD4+, and CD8+ cells (Fig. 5B, G2 cells). In nonimmunized animals, the percentage and the absolute number of retinal microglia (CD45intCD11b+) was comparable between WT (0.19% ± 0.04%, 5030 ± 1044 cells/retina) and DKO (0.20% ± 0.02%, 4392 ± 1229 cells/retina) retinas (Fig. 5C). The number of microglia increased significantly in EAU retinas at days 25 and 90 p.i. (Fig. 5C), suggesting in situ proliferation of retinal microglia during inflammation.27 Many more microglial cells were detected in WT EAU retinas compared with DKO EAU retinas (Fig. 5C). The proportion of CD45hi cells was negligible in retinas from nonimmunized WT and DKO mice, but massively increased during EAU. Wild-type EAU retinas contained 8.4 times more infiltrating cells than DKO retinas at day 25 p.i. and 1.3 times more at day 90 p.i. (Fig. 5D).

Microglia and infiltrating cells in the inflamed retina at different stages of EAU in WT and DKO mice. Retinas from nonimmunized, days 25 and 90 p.i. WT and DKO mice were collected. Single-cell suspension was prepared (see Methods section) and stained for CD45, CD11b, Gr-1, Ly6G, CD4, and CD8. Samples were analyzed by flow cytometry. (A) Dot-plot showing CD11b+CD45int (G1) and CD45hi (G2) cells. (B) G1 (CD11b+CD45int) cells were Ly6G−, Gr-1−, CD4−, and CD8− and were considered as microglia. The G2 (CD45hi) cells were heterogeneous, including Ly6G+, Gr-1+, CD4+, and CD8+ cells, and were considered as infiltrating leukocytes. (C) The percentage and number of retinal microglia cells in different stages of EAU in WT and DKO mice. (D) The percentage and number of retinal infiltrating cells in different stages of EAU in WT and DKO mice. Data are presented as mean ± SEM. n = 3. *P < 0.05 compared with WT mice of the same time point, unpaired Student's t-test.

Figure 5

Microglia and infiltrating cells in the inflamed retina at different stages of EAU in WT and DKO mice. Retinas from nonimmunized, days 25 and 90 p.i. WT and DKO mice were collected. Single-cell suspension was prepared (see Methods section) and stained for CD45, CD11b, Gr-1, Ly6G, CD4, and CD8. Samples were analyzed by flow cytometry. (A) Dot-plot showing CD11b+CD45int (G1) and CD45hi (G2) cells. (B) G1 (CD11b+CD45int) cells were Ly6G−, Gr-1−, CD4−, and CD8− and were considered as microglia. The G2 (CD45hi) cells were heterogeneous, including Ly6G+, Gr-1+, CD4+, and CD8+ cells, and were considered as infiltrating leukocytes. (C) The percentage and number of retinal microglia cells in different stages of EAU in WT and DKO mice. (D) The percentage and number of retinal infiltrating cells in different stages of EAU in WT and DKO mice. Data are presented as mean ± SEM. n = 3. *P < 0.05 compared with WT mice of the same time point, unpaired Student's t-test.

Phenotype analysis showed that both myeloid-derived cells, including CD11b+F4/80+ macrophages (Fig. 6A), CD11b+Gr-1+Ly6G+ neutrophils (Fig. 6B), CD11b+Gr-1+Ly6G− MDSCs (Fig. 6B), CD11c+ dendritic cells (DCs; Fig. 6C), and lymphoid cells, including CD4+ and CD8+ T cells (Fig. 6D) and B220+ B cells (Fig. 6E) were present in both WT and DKO EAU retinas. Lymphoid:myeloid ratio was 0.66 at day 25 p.i., and 0.96 at day 90 p.i. in WT mice. In DKO mice, the ratio was 0.92 and 2.42 at days 25 and 90 p.i. respectively (Fig. 6F). Since the total number of infiltrating cells was lower in DKO mice, the results suggest that the recruitment of myeloid-derived cells to the inflamed retina is impaired in DKO mice, particularly at day 90 p.i.

To further understand the retinal immune response in DKO EAU mice, we examined the gene expression of various chemokines and cytokines in the retinas of WT and DKO mice at different stages of EAU by real-time RT-PCR.

Following EAU induction, the expression of two CC chemokines (Ccl2 and Ccl5) was massively increased (>200-fold) at day 25 p.i., and the expression remained at high levels (>150-fold) at days 60 and 90 p.i. in WT retinas (Fig. 7A). Double knockout mouse retinas also express high levels of CCL5 mRNA, although it was slightly lower than WT mice at day 25 p.i. The CCL2 mRNA was not detected in DKO mouse retina (Fig. 7A). The expression of CXC chemokines (Cxcl12 and Cxcl10) was markedly upregulated after EAU induction in WT mice, but only mildly increased in DKO mice (Fig. 7A). The expression of Cx3cl1 was slightly increased (less than 5-fold) in the retinas in both WT and DKO mice during EAU.

In WT EAU retina, the expressions of Tnfa, Il1b, and inducible nitric oxide synthase (iNOS or NOS2) increased significantly at day 25 p.i. and declined during chronic stages (days 60 and 80 p.i.; Fig. 7B). Although the expression of Tnfa was also increased in DKO EAU retina, the expression level was lower than that in WT EAU retina at each time point. The expression of iNOS and Il1b between DKO and WT retinas did not differ at any time point (Fig. 7B). The expression of Vegfa and Arg-1 in WT retinas was increased during the chronic stages of EAU, but only a mild increment of Vegfa was observed in DKO retinas. At day 90 p.i., DKO retinas expressed a significantly lower level of Arg-1 compared with WT retinas (Fig. 7B).

Discussion

Previous studies have shown that the deletion of CCL2, CCR2, or CX3CR1 does not affect the severity of EAU.16,18 In this study, we found that the combined deletion of CCL2 and CX3CR1 resulted in reduced retinal inflammation at the acute but not the chronic stage of EAU. Although the severity of chronic inflammation was comparable to WT mice, retinal structural damage and angiogenesis were markedly reduced in DKO mice, which appears to be related to impaired recruitment of macrophages and MDSCs to the inflamed retina.

After immunization, DKO and WT mice had similar kinetic profiles of circulating immune cells (except F4/80+ macrophages, which were lower in DKO mice), suggesting that these mice had comparable levels of systemic immune response to IRBP immunization. The F4/80 is expressed predominately by tissue macrophages, although low levels of F4/80 also may be expressed by circulating monocytes (precursor of macrophages).28 During inflammation, monocytes are recruited to the inflamed tissue and differentiate into F4/80+ macrophages. The CCL2-CCR2 and CX3CL1-CX3CR1 pathways play critical roles in monocyte trafficking to site of inflammation. It has been shown that inflammatory macrophages may not die locally, rather they migrate back to the circulation and end in secondary lymphoid organs where they are cleared by unknown mechanisms.29 The reduced F4/80+ macrophages in the blood and spleen of DKO EAU mice may result from decreased tissue macrophage supply.

At the acute stages of EAU, the ratio of lymphocytes:myeloid cells was slightly higher in DKO mice compared with WT mice. However, DKO retinas had more neutrophils and fewer macrophages compared with WT retinas (Fig. 6), suggesting neutrophil-dominated retinal inflammation at this stage. A previous study showed that retinal inflammation in CCL2 KO EAU mice was mediated predominately by neutrophils.16 However, unlike the DKO mice, CCL2 KO EAU mice had similar levels of retinal inflammation and tissue damage compared with WT mice16 (Supplementary Fig. S1). Tumor necrosis factor-α and nitric oxide are known to be the main mediators responsible for tissue damage at the acute stage of EAU.30,31 Infiltrating macrophages, in particular M1 subset, are the main source of TNF-α during acute inflammation. Other cells, such as neutrophils and CD4+ T cells, also can produce this cytokine, but their ability is relatively low.32 Tumor necrosis factor-α expression was significantly lower in DKO mouse retina compared with that in WT mice. In the DKO mice, the trafficking of both CCR2+ and CX3CR1+ monocytes is impaired, whereas deletion of CCL2 affects only CCR2+ monocyte trafficking. Our results suggest that CX3CR1+ monocyte-derived macrophages may contribute, at least in part, to the severity of inflammation and tissue damage in EAU.

At the chronic stages of EAU (i.e., day 60–90 p.i.), even though the severity of inflammation did not differ between WT and DKO mice, retinal structural damage, and in particular angiogenesis, is massively reduced in DKO mice. This may be partially explained by less inflammatory damage at the acute stage of EAU; however, the type of infiltrating immune cells may be more important. T cells (CD4+ and CD8+), B cells, macrophages, DCs, and MDSCs, but not neutrophils, were detected in significant amounts in the inflamed retina in both WT and DKO mice at day 90 p.i. This suggests that neutrophils may play an insignificant role in chronic EAU. Accumulation of CD8+ memory T cells has been observed in chronic EAU and they are known to express inhibitory receptors, such as PD-1, that can limit inflammation.33 However, the amount of retinal infiltrating CD8+ T cells was similar between WT and DKO mice in chronic EAU. The MDSCs and F4/80+ macrophages constitute 10% and 19% of retinal infiltrating cells in WT mice but only 3% and 8% in DKO mice at day 90 p.i. CD4+ T cells constitute 51% of retinal infiltrating cells in DKO EAU mice. The MDSCs play an important role in suppressing inflammation34 and the number is increased in late stages of EAU.35 The reduced MDSCs in DKO mice may explain the sustained CD4+ T-cell infiltration and persistent inflammation in the mice. Whereas the reduced macrophage infiltration may account for less tissue damage and angiogenesis (see below discussion). Our results may support the role of the CCL2-CCR2 pathway in MDSC trafficking, reported previously by Sawanobori and colleagues.36 The involvement of the CX3CL1-CX3CR1 pathway in MDSC trafficking remains to be elucidated.

Tissue structural damage and angiogenesis at the late stages of inflammation is related to postinflammation tissue repair and remodeling. Despite comparable levels of chronic inflammation for more than 2 months (i.e., from day 25 to 90 p.i.) in WT and DKO mice, retinal structural damage and angiogenesis were significantly reduced in DKO mice. The expression levels of VEGF in DKO EAU retinas were only slightly lower than those in WT retina at days 60 and 90 p.i., suggesting that other factors might be involved in the chronic inflammation-mediated retinal angiogenesis. Macrophages, in particular M2-type wound-healing cells, are known to play an important role in tissue repair/remodeling. Previously we showed that Arginase-1+ macrophages are increased in late stages (i.e., angiogenic stage) of EAU.17 Whether the wound-healing macrophages originated from CCR2+ or CX3CR1+ monocytes or both remains unknown. In this study, we further found that retinal angiogenesis was reduced by 75% in CCL2 KO EAU mice and 45% in CX3CR1 KO EAU mice. The deletion of both CCL2 and CX3CR1 resulted in a 93% reduction in EAU-induced retinal angiogenesis. Our result suggests that the CX3CR1+ macrophages may work together with the CCR2+ macrophages in postinflammation retinal repair and remodeling, although CCR2+ macrophages may play a dominating role.

In summary, we show in this study that the deletion of CCL2 and CX3CR1 in mice results in neutrophil-dominated acute inflammation and T-lymphocyte–dominated chronic inflammation in the EAU model. Neutrophil-dominated retinal inflammation is less destructive compared with classic macrophage-dominated inflammation, and postinflammation tissue repair and wound healing is impaired in T-cell–dominated chronic inflammation. Our study highlights the role of both CCR2+ and CX3CR1+ macrophages in retinal tissue damage and angiogenesis in EAU.

Acknowledgments

The authors thank Aisling Lynch for helping with English expression.

Supported by the National Eye Research Centre (SCAID061) and Fight for Sight (1361/2).

Flow cytometric analysis of immune cells in the blood and spleen of WT and DKO mice. Leukocytes from blood and spleen of WT and DKO mice were analyzed by flow cytometry for the presence of various immune cells. (A) Bar graph showing the percentages of various immune cells in the blood in nonimmunized WT and DKO mice. (B) The percentage of different subsets of immune cells in the blood at different stages of EAU in WT and DKO mice. (C) Bar graph showing the percentages of various immune cells in the splenic in nonimmunized WT and DKO mice. (D) the percentage of different subsets of immune cells in the spleen at different stages of EAU. Data were presented as mean ± SEM. n = 3. *P < 0.05, **P < 0.01 compared with nonimmunized mice. +P < 0.05 , ++P < 0.01 compared with WT mice of the same time point.

Figure 4

Flow cytometric analysis of immune cells in the blood and spleen of WT and DKO mice. Leukocytes from blood and spleen of WT and DKO mice were analyzed by flow cytometry for the presence of various immune cells. (A) Bar graph showing the percentages of various immune cells in the blood in nonimmunized WT and DKO mice. (B) The percentage of different subsets of immune cells in the blood at different stages of EAU in WT and DKO mice. (C) Bar graph showing the percentages of various immune cells in the splenic in nonimmunized WT and DKO mice. (D) the percentage of different subsets of immune cells in the spleen at different stages of EAU. Data were presented as mean ± SEM. n = 3. *P < 0.05, **P < 0.01 compared with nonimmunized mice. +P < 0.05 , ++P < 0.01 compared with WT mice of the same time point.

Microglia and infiltrating cells in the inflamed retina at different stages of EAU in WT and DKO mice. Retinas from nonimmunized, days 25 and 90 p.i. WT and DKO mice were collected. Single-cell suspension was prepared (see Methods section) and stained for CD45, CD11b, Gr-1, Ly6G, CD4, and CD8. Samples were analyzed by flow cytometry. (A) Dot-plot showing CD11b+CD45int (G1) and CD45hi (G2) cells. (B) G1 (CD11b+CD45int) cells were Ly6G−, Gr-1−, CD4−, and CD8− and were considered as microglia. The G2 (CD45hi) cells were heterogeneous, including Ly6G+, Gr-1+, CD4+, and CD8+ cells, and were considered as infiltrating leukocytes. (C) The percentage and number of retinal microglia cells in different stages of EAU in WT and DKO mice. (D) The percentage and number of retinal infiltrating cells in different stages of EAU in WT and DKO mice. Data are presented as mean ± SEM. n = 3. *P < 0.05 compared with WT mice of the same time point, unpaired Student's t-test.

Figure 5

Microglia and infiltrating cells in the inflamed retina at different stages of EAU in WT and DKO mice. Retinas from nonimmunized, days 25 and 90 p.i. WT and DKO mice were collected. Single-cell suspension was prepared (see Methods section) and stained for CD45, CD11b, Gr-1, Ly6G, CD4, and CD8. Samples were analyzed by flow cytometry. (A) Dot-plot showing CD11b+CD45int (G1) and CD45hi (G2) cells. (B) G1 (CD11b+CD45int) cells were Ly6G−, Gr-1−, CD4−, and CD8− and were considered as microglia. The G2 (CD45hi) cells were heterogeneous, including Ly6G+, Gr-1+, CD4+, and CD8+ cells, and were considered as infiltrating leukocytes. (C) The percentage and number of retinal microglia cells in different stages of EAU in WT and DKO mice. (D) The percentage and number of retinal infiltrating cells in different stages of EAU in WT and DKO mice. Data are presented as mean ± SEM. n = 3. *P < 0.05 compared with WT mice of the same time point, unpaired Student's t-test.